Method of separating poly-3-hydroxyalkanoic acid

ABSTRACT

The invention aims at providing a method of producing a poly-3-hydroxyalkanoic acid, which comprises carrying out a physical disruption treatment of a suspension of poly-3-hydroxyalkanoic acid-containing microbial cells with adding an alkali thereto either continuously or intermittently and, thereafter, separating the poly-3-hydroxyalkanoic acid.

TECHNICAL FIELD

The present invention relates to a method for the separation andpurification of poly-3-hydroxyalkanoic acids from microbial cells.

BACKGROUND ART

Poly-3-hydroxyalkanoic acids (hereinafter sometimes referred tocollectively as PHA) are the thermoplastic polyesters which areelaborated and accumulated as energy storage substances by a variety ofmicroorganisms and have biodegradability. In these days waste plasticsare disposed of by incineration or burial but these methods of disposalare causative of global warming and ground loosening of reclaimed lands,among other disadvantages. Therefore, with the growing public awarenessof the importance of plastics recycling, ways and means for systematizedrecycling are being explored. However, uses amenable to such recyclingare limited and actually the disposal load of waste plastics cannot becompletely liquidated by said incineration, burial, and recycling butrather a large proportion of the disposal load is not disposed of butsimply left exposed to the elements. There is accordingly a mountinginterest in PHA and other biodegradable plastics which, after disposal,would be incorporated into the natural cycle of matter and degradationproducts of which would not exert ecologically deleterious influences,and their practical utilization are highly desired. Particularly the PHAwhich microorganisms elaborate and accumulate in their cells is taken upin the carbon cycle of the natural kingdom and it is, therefore,predicted that it will not have any appreciable adverse effects on theecosystem. In the field of medical treatment, too, it is consideredpossible to use PHA as an implant material which does not requirerecovery or a vehicle for drug delivery.

Since the PHA elaborated by microorganisms usually form granules and isaccumulated intracellularly, exploitation of PHA as a plastic requires aprocedure for separating it from microbial cells. The known technologyfor the separation and purification of PHA from microbial cells can beroughly classified into the technology which comprises extracting PHAfrom the cells with an organic solvent in which PHA is soluble and thetechnology which comprises removing the cell components other than PHAby cell disruption or solubilization.

Referring to the separation and purification technology of PHA involvingextraction with an organic solvent, the extraction technique utilizing ahalogen-containing hydrocarbon, such as 1,2-dichloroethane orchloroform, as the solvent in which PHA is,soluble is known (JapaneseKokai Publication Sho-55-118394, Japanese Kokai PublicationSho-57-65193). However, since these halogen-containing hydrocarbons arehydrophobic solvents, a pre-extraction procedure, such as drying thecells in advance or otherwise, allowing the solvent to directly contactthe intracellular PHA is required. Moreover, in such a technology,dissolving PHA at a practically useful concentration (for example, 5%)or higher gives only an extract which is so highly viscous that itinvolves considerable difficulties in separating the undissolvedresidues of microbial cells from the PHA-containing solvent layer.Furthermore, in order that PHA may be reprecipitated from the solventlayer at a high recovery, some PHA-insoluble solvent, such as methanolor hexane, need to be used in a large quantity, e.g. 4 to 5 volumesbased on the solvent layer, and thus a vessel of large capacity isrequired for reprecipitation. In addition, the necessary quantity ofsolvents is so large that both the solvent recovery cost and the cost oflost solvents are enormous. Furthermore since the use of organohalogencompounds tends to be limited these days for protection of theenvironment, industrial application of this technology has manyobstacles to surmount.

Under the circumstances, there has been proposed an extractiontechnology using a solvent which is not only capable of dissolving PHAbut also miscible with water, for example a hydrophilic solvent such asdioxane (Japanese Kokai Publication Sho-63-198991), propanediol(Japanese Kokai Publication Hei-02-69187), or tetrahydrofuran (JapaneseKokai Publication Hei-07-79788). These methods appear to be favorablepartly because PHA can be extracted not only from dry cells but alsofrom wet cells and partly because precipitates of PHA can be obtained bymere cooling of the solvent layer separated from the microbial cellresidues. However, even with these methods, the problem of highviscosity of the PHA-containing solvent layer remains to be solved. Inaddition, while heating is required for enhancing the extractionefficiency, the heating in the presence of water unavoidably results ina decrease in molecular weight due to hydrolysis of PHA and a poorrecovery of PHA.

On the other hand, as the technology of removing the cell componentsother than PHA by solubilization for separation of PHA, J. Gen.Microbiology, 19, 198-209 (1958) describes a technology which comprisestreating a suspension of microbial cells with sodium hypochlorite tosolubilize cell components other than PHA and recovering PHA. Thistechnology is simple process-wise but the necessity to use a largeamount of sodium hypochlorite is a factor leading to a high productioncost. Moreover, in view of the marked decrease in molecular weight ofPHA and the appreciable amount of chlorine left behind in PHA, thistechnology is not considered to be suitable for practical use. JapaneseKokoku Publication Hei-04-61638 describes a process for separating PHAwhich comprises subjecting a suspension of PHA-containing microbialcells to a heat treatment at a temperature of 100° C. or higher todisrupt the cellular architecture and, then, subjecting the disruptedcells to a combination treatment with a protease and either aphospholipase or hydrogen peroxide to solubilize the cell componentsother than PHA. This technology is disadvantageous in that because theheat treatment induces denaturation and insolubilization of the protein,the load of subsequent protease treatment is increased and that theprocess involves many steps and is complicated.

As a technology for disrupting PHA-containing microbial cells, therealso has been proposed a method which comprises treating microbial cellswith a surfactant, decomposing the nucleic acids released from the cellswith hydrogen peroxide, and separating PHA (Japanese Kohyo PublicationHei-08-502415) but the waste liquor containing the surfactant develops acopious foam and, in addition, has a high BOD load value. From thesepoints of view, the use of a surfactant is objectionable for productionon a commercial scale.

There has also been proposed a technology for separating PHA whichcomprises disrupting PHA-containing microbial cells with a high-pressurehomogenizer (Japanese Kokai Publication Hei-07-177894 and Japanese KokaiPublication Hei-07-31488). However, this technology has the drawbackthat although a suspension of microbial cells is subjected to ahigh-pressure treatment at least 3 times, or 10 times at elevatedtemperature depending on cases, the purity of PHA that can be attainedis as low as about 65 to 89%. There has also been proposed a technologyfor separating PHA which comprises adding an alkali to a suspension ofPHA-containing microbial cells, heating the suspension, and disruptingthe cells (Japanese Kokai Publication Hei-07-31487). However, thistechnology is disadvantageous in that the purity of the product polymerthat can be attained is as low as 75.1 to 80.5% and that if the level ofaddition of the alkali be raised to improve the yield, the molecularweight of the polymer would be decreased. Several techniques forcarrying out physical disruption after addition of an alkali have beenproposed (Bioseparation, 2, 95-105, 1991, Japanese Kokai PublicationHei-07-31489) but since the alkali treatment alone results in theextracellular release of only a small amount of cell components and someof such cell components are retained in the PHA fraction even aftersubsequent high-pressure disruption treatment, these techniques areinvariably inefficient. Thus, PHA of high purity cannot be separatedunless the microbial cell suspension is subjected to at least 5 cyclesof high-pressure treatment and even then the purity of PHA is as low asabout 77 to 85%. The technologoy involving addition of an alkali has anadditional drawback; generally the cell components released frommicrobial cells, particularly nucleic acids, increase the viscosity ofthe cell suspension to make subsequent processing difficult.

There has also been proposed a technology in which a suspension ofPHA-containing microbial cells is adjusted to an acidity lower than pH 2and PHA is separated at a temperature not below 50° C. (Japanese KokaiPublication Hei-11-266891). However, this technology is disadvantageousin that the treatment under the strongly acidic condition below pH 2 isundesirable for production on a commercial scale, that the acidtreatment must be followed by adjustment to the alkaline side forenhanced purity but this entails massive salt formation, and that themolecular weight of the product PHA is decreased from 2,470,000 to about1,000,000.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the abovedisadvantages of the prior art and accordingly provide a technology ofseparating and purifying PHA which is capable of removing the cellcomponents other than PHA with good efficiency from PHA-containingmicrobial cells and giving PHA of high purity in high yields in only afew steps without incurring any serious decrease in molecular weight ofPHA.

The inventors of the present invention explored in earnest for acommercially useful PHA production technology. As a result they found(i) that when a physical disruption treatment of a suspension ofPHA-containing microbial cells is carried out with adding an alkalithereto either continuously or intermittently, the viscosity elevationof the suspension due to the release of cell components other than PHAfrom the cells can be prevented, (ii) that pH control of the suspensionis rendered feasible by the above prevention of viscosity elevation ofthe suspension, and (iii) that by this feasiblity of suspension pHcontrol, the treatment at a low alkali concentration can be madepossible with the consequent advantage that PHA of high purity can beseparated without incurring a marked decrease in molecular weight. Thepresent invention has been developed on the basis of the above findings.

The present invention, therefore, relates to a method of producing apoly-3-hydroxyalkanoic acid,

which comprises carrying out a physical disruption treatment of asuspension of poly-3-hydroxyalkanoic acid-containing microbial cellswith adding an alkali thereto and, thereafter, separating thepoly-3-hydroxyalkanoic acid.

In one preferred mode of practicing the invention, the present inventionrelates to the method,

wherein said addition of an alkali is carried out with controlling thepH of the suspension, more preferably the PH of the suspension between 9and 13.5. It should also be understood that said physical disruptiontreatment of the suspension is preferably carried out under stirring ofsaid suspension. Furthermore, said physical disruption treatment of thesuspension is preferably carried out at the temperature not less than20° C. and below 40° C.

In another preferred mode of practicing the invention, the presentinvention relates to the method,

wherein the poly-3-hydroxyalkanoic acid is a copolymer comprising ofD-3-hydroxyhexanoate (3HH) and one or more other 3-hydroxyalkanoicacids. More preferably, the present invention relates to the method,

wherein the poly-3-hydroxyalkanoic acid is a binary copolymer comprisingof D-3-hydroxybutyrate (3HB) and D-3-hydroxyhexanoate (3HH) or a ternarycopolymer comprising of D-3-hydroxybutyrate (3HB), D-3-hydroxyvalerate(3HV), and D-3-hydroxyhexanoate (3HH).

In a still another preferred mode, the present invention relates to themethod,

wherein the poly-3-hydroxyalkanoic acid-containing microbial cells arecells of Aeromonas caviae or cells of a strain of microorganismtransformed by a poly-3-hydroxyalkanoic acid synthase group gene derivedfrom Aeromonas caviae.

The present invention is now described in detail.

DETAILED DESCRIPTION OF THE INVENTION

The microorganism for use in the present invention is not particularlyrestricted provided that it is a microorganism containing PHA asintracellularly accumulated. For example, microorganisms of the genusAlcaligenes, such as A. lipolytica, A. eutrophus, A. latus, etc.; thoseof the genus Pseudomonas; those of the genus Bacillus, those of thegenus Azotobacter; those of the genus Nocardia; and those of the genusAeromonas can be mentioned. Particularly preferred are strains ofAlcaligenes caviae, and further are strains of Alcaligenes eutrophusAC32 transformed by a PHA synthase group gene derived therefrom(deposited on Budapest Treaty, international depositary authority:National Institute of Advanced Industrial Science and TechnologyInternational Patent Organism Depositary, 1-3 Higashi 1 chome,Tsukuba-shi, Ibaraki-ken, Japan, date of transfer: Aug. 7, 1997,Accession No. FERM BP-6038, as transferred from FERM P-15786 originallydeposited Aug. 12, 1996 (J. Bacteriol., 179, 4821-4830 (1997)). In thepresent invention, such a strain of microorganism is cultured undersuitable conditions to let it accumulate PHA intracellularly and itscells are used. The cultural method is not particularly restricted butthe known method described in Japanese Kokai Publication Hei-05-93049,among others, can for example be employed.

The term “PHA” as used in this specification is a generic term meaningany and all polymers of hydroxyalkanoic acids. Although thehydroxyalkanoic acid units of such polymers are not particularlyrestricted, a homopolymer comprising of D-3-hydroxybutyrate (3HB), acopolymer of 3HB and one or more other 3-hydroxyalkanoic acids, and acopolymer of various 3-hydroxyalkanoic acids inclusive ofD-3-hydroxyhexanoate (3HH) can be mentioned by way of example.Particularly preferred from the standpoint of physical characteristicsof the product polyester is the polymer containing 3HH as a monomericunit, for example a binary copolymer comprising of 3HB and 3HH(Macromolecules, 28, 4822-4828 (1995)) or a ternary copolymer comprisingof 3HB, D-3-hydroxyvalerate (3HV), and 3HH (Japanese Patent No. 277757,Japanese Kokai Publication Hei-08-289797). The compositional ratio ofthe monomer units constituting a binary copolymer comprising of 3HB and3HH is not particularly restricted but copolymers containing 1 to 99 mol% of the 3HH unit are suitable. The compositional ratio of the monomerunits constituting a ternary copolymer comprising of 3HB, 3HV, and 3HHis not particularly restricted, either, but copolymers containing 1 to95 mol % of the 3HB unit, 1 to 96 mol % of the 3HV unit, and 1 to 30 mol% of the 3HH unit are preferred.

The PHA content of the microbial cells to be treated is preferably ashigh as possible, of course. In the treatment on a commercial scale, thePHA content of dry cells is preferably not less than 20 weight %, andwhen the alkali treatment, physical disruption treatment, separationprocedure, and purity of the separated polymer, among other factors, aretaken into consideration, the particularly preferred PHA content is notless than 50 weight %.

The term “a suspension of microbial cells” as used in this specificationmeans a culture medium available on completion of culture as is or anaqueous suspension of the cells harvested from a culture medium bycentrifugation or the like technique. The concentration of cells in thesuspension on a dry cell basis is preferably not more than 500 g/L, morepreferably not more than 300 g/L.

The alkali for use in the practice of the invention is not particularlyrestricted provided that the suspension pH may be controlled within theherein-defined range, and includes alkali metal hydroxides such assodium hydroxide, potassium hydroxide, lithium hydroxide, etc.; alkalimetal carbonates such as sodium carbonate, potassium carbonate, etc.;alkali metal hydrogen carbonates such as sodium hydrogen carbonate,potassium hydrogen carbonate, etc.; organic acid alkali metal salts suchas sodium acetate, potassium acetate, etc.; alkali metal borates such asborax etc.; alkali metal phosphates such as trisodium phosphate,disodium hydrogen phosphate, tripotassium phosphate, dipotassiumhydrogen phosphate, etc., and aqueous ammonia, among others. Amongthese, sodium hydroxide, sodium carbonate, and potassium hydroxide arepreferred in terms of suitability for commercial production and in costterms. The alkali may be added directly but is preferably added in theform of an aqueous solution.

The physical disruption treatment in the present invention includes notonly sonication but also disruption with an emulsification-dispersionmachine, a high-pressure homogenizer, a mill or the like. Thehigh-pressure homogenizer referred to above is not particularlyrestricted but may include Manton-Gaulin manufactured by APV Gaulin,Germany, Mini-Lab manufactured by APV Rannie, Demmark, andMicrofluidizer manufactured by Microfluidics, USA, among others. Themill referred to above is not particularly restricted but includesDYNO-Mill manufactured by Willy A. Bachofen, Switzerland, to mention anexample. The emulsification-dispersion machine referred to above is notparticularly restricted, either, but includes SILVERSON MIXERmanufactured by Silverson Machines, Inc., England, Clearmix manufacturedby M-TECHNIQUE, Japan, and Ebara Milder manufactured by EbaraCorporation, Japan, among others. These machines are not exclusivechoices but any machine capable of causing efficient disruption ofnucleic acids, which are released from cells in the alkali treatment andotherwise mainly play a part in causing suspension viscosity elevation,and, at the same time, capable of effecting sufficient dispersion ofinsoluble substance other than the objective polymer, such as the cellwall, cell membrane, and insoluble protein, can be employed.Furthermore, the purity of the polymer can be enhanced by operating twoor more of the above-mentioned kinds of disrupting machines concurrentlyor in succession.

The physical disruption treatment referred to above can be carried outunder stirring of said suspension. Stirring means which can be used isnot particularly restricted but may be any means capable of producingthe ordinary mechanical stirring effect.

In the present invention, the physical disruption treatment of asuspension of PHA-containing microbial cells is carried out with addingthe alkali either continuously or intermittently. By this alkalitreatment, insoluble substances such as nucleic acids, cell wall, cellmembrane and insoluble protein are released along with PHA from themicrobial cells. As the physical disruption treatment is carried outsimultaneously, the cells are completely disrupted and the releasedconstituents are micronized to prevent viscosity elevation and promotethe alkali solubilization of insoluble substances, thus contributing toenhanced PHA yields.

In the present invention, said physical disruption treatment and alkaliaddition may be carried out concurrently or the physical disruptiontreatment may be started in advance of the start of alkali addition. Asa further alternative, said physical disruption treatment and alkaliaddition may be carried out in an alternating fashion, or following saidphysical disruption treatment and alkali addition, the physicaldisruption treatment alone may be further continued.

In the present invention, it is preferable to control the pH of saidsuspension at the time of alkali addition. The control target pH valueis preferably not less than pH 9, more preferably not less than pH 10.Moreover, the pH is controlled still more preferably at not more than pH13.5, further more preferably not more than pH 13. If the pH exceeds13.5, marked decomposition of PHA may take place. If the pH is less than9, the PHA separation effect tends to be sacrificed in some cases. Thelatitude of pH control is preferably within ±1 of the set value, morepreferably within ±0.5 of the set value.

In the practice of the invention, the addition of the alkali ispreferably carried out either continuously or intermittently withcontrolling the pH of said suspension.

The inventors of the present invention found empirically that, in theseparation and purification of PHA from microbial cells, the addition ofthe whole amount of alkali at one time as in the prior art results inthe exposure of PHA to a high concentration of alkali immediately afteraddition to cause a decrease in molecular weight of PHA and lead to aprogressive depression of pH resulting from the consumption of thealkali as the reaction proceeds so that the efficient extraction cannotbe consistently carried through. Moreover, in the technology involvingaddition of a predetermined amount of alkali to microbial cells, theculture medium components contained in the cell suspension, if they areacidic substances, react with the alkali or give rise to a bufferedcondition, thus failing to achieve the expected result. In contrast,according to the preferred technology of the invention, in which theaddition of the alkali is carried out either continuously orintermittently to control the pH on the alkaline side, the insolublesubstance can be effectively dissolved to realize an effectiveseparation of PHA. Moreover, since the level of addition of the alkaliis controlled not according to the absolute amount of alkali to be addedbut according to the pH, reproducible results can be obtained withoutbeing affected by the cultural conditions used, the time followingculture till cell disruption, or the secondary substances occurring inthe cell suspension.

From the standpoint of pH control, too, it is necessary that thephysical disruption treatment of the suspension should be carried outwith adding an alkali to the suspension. If the addition of an alkali isnot carried out in the physical disruption treatment, the viscosity ofthe cell suspension is increased as mentioned above to interfere withstirring so that the pH cannot be controlled. As a consequence, aconcentration distribution of the alkali added takes place with theconsequent local elevation of alkali concentration inducing a decreasein molecular weight of PHA.

In the present invention, unlike in the prior art, physical disruptiontreatment and alkali addition need not be carried out at hightemperature for separating PHA from PHA-containing microbial cells.Rather, a high-temperature treatment under alkaline conditions should beavoided, for otherwise it would induce a decrease in molecular weight ofPHA. The preferred temperature for physical disruption treatment andalkali addition in the present invention is not over 50° C., morepreferably not over 40° C., still more preferably below 40° C. The lowerlimit is preferably 20° C., more preferably 25° C.

FIGS. 1(a) and (b) are schematic diagrams showing the exemplaryequipment for microbial cell disruption for use in the separation andpurification of PHA according to the invention. Of course, the mode ofcarrying out the invention is by no means limited to the one using theillustrated equipment.

The reference numeral 1 generally indicates the microbial celldisrupting equipment according to the invention. The reference numeral6, in FIGS. 1(a) and (b), indicates a pH control agent strage tankadapted to hold a reserve of the alkali, and the pH control agent inthis pH control agent strage tank 6 is fed by a pump 4 to a celldisruption tank 11 through a pipeline 5 to adjust the pH of a microbialsuspension in the cell disruption tank 11. This cell disruption tank 11is equipped with a stirring means 2 for uniformly stirring and mixingthe pH control agent from the pH control agent strage tank 6 with themicrobial cell suspension in the cell disruption tank 11. The same celldisruption tank 11 is further equipped with a pH detection-control meansconsisting of a pH meter 7 and a pH sensor-controller for detecting thepH of the microbial cell suspension in the cell disruption tank 11 andcontrolling the rate of feed of the pH control agent by said pump 4 sothat a predetermined pH level may be established.

Referring to FIG. 1(a), the microbial cell suspension in the celldisruption tank 11 is fed by a pump 10 to a disrupting device 9 which isadapted to efficiently disrupt the nucleic acids otherwise causative ofviscosity elevation as released from the microbial cells and feed thecells to the cell disruption tank 11 via a pipeline 8. In thisarrangement, the viscosity of the cell suspension in the cell disruptiontank 11 is held low and the cell suspension is homogenized by thestirring means 2, thus making it possible to strictly control the pH ofthe cell suspension.

Referring to FIG. 1(b), a disrupting device 12 is equipped within thecell disruption tank 11 so that the nucleic acids otherwise causative ofviscosity elevation as released from the microbial cells may beefficiently disrupted by said disrupting device 12 within said celldisruption tank 11. Moreover, in the case where the disrupting device 12has both the function to disrupt the nucleic acids and the function touniformly stir the cell suspension, the stirring means 2 illustrated inthe diagram (b) may be omitted.

PHA as obtained by the method of the invention is of high purity butdepending on the intended use, the purity of this polymer may be furtherimproved by the known purification technology using, for example, alytic enzyme such as lysozyme (Japanese Kokoku PublicationHei-04-61638), a protease such as trypsin or pronase (Japanese KokaiPublication Hei-05-336982), or a peroxide such as hydrogen peroxide(Japanese Kohyo Publication Hei-08-502415).

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1(a) and (b) are schematic diagrams showing examples of theequipment for disrupting microbial cells in practicing the method ofproducing PHA according to the invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The strain of microorganism used in this example is Alcaligeneseutrophus AC32 (detailed information for accession are givenhereinabove) transformed by a PHA synthase group gene derived fromAeromonas caviae. This strain was cultured in accordance with theprotocol given in J. Bacteriol., 179, 4821-4830 (1997) to harvestbacterial cells containing about 60 wt % ofpoly(D-3-hydroxybutyrate-co-D-3-hydroxyhexanoate) [hereinafter sometimesreferred to briefly as p(3HB-co-3HH)] having an average molecular weightof 1,000,000. The culture medium thus obtained was centrifuged (5,000rpm, 10 min) to separate the cells and this pasty cellular fraction wasdiluted with water to prepare an aqueous suspension of 50 g cells/Lconcentration. This aqueous suspension was subjected to the followingExamples, although the invention is by no means limited to theparticular Examples.

The purity of the p(3HB-co-3HH) separated from the cells has determiendas follows. A precipitate, 10 mg, as separated from the cells wasdissolved in 1 ml of chloroform and treated with 0.85 ml of methanol and0.25 ml of concentrated sulfuric acid at 100° C. for 140 minutes. Aftercooling, 0.5 ml of a saturated aqueous solution of ammonium sulfate wasadded and the mixture was stirred vigorously and, then, allowed tostand. The bottom layer was analyzed by capillary gas chromatography todetermine the purity of the separated p(3HB-co-3HH).

The molecular weight of the p(3HB-co-3HH) separated from the bacterialcells was determined as follows. The precipitate (10 mg) separated fromthe bacterial cells was dissolved in 1 ml of chloroform and the solutionwas filtered to remove the insoluble substance. The filtrate wasanalyzed with SHIMADZU Corporation's GPC System fitted with TosohCorporation's TSK-GEL GMHXL (7.8×300 mm, two columns connected inseries) using chloroform as the mobile phase.

EXAMPLE 1

Using the p(3HB-co-3HH)-containing bacterial cells, 500 mL of a cellsuspension was prepared and this suspension was placed in a 1 L reactionvessel equipped with a pH electrode and SILVERSON MIXER and incubated at35° C. The pH electrode was connected to Lab Controller MDL-6Cmanufactured by B. E. Marubishi Co., Ltd. and the operation parameterswere so set that when the pH of the suspension had dropped below a setvalue, a peristaltic pump would be actuated to deliver an aqueoussolution of sodium hydroxide into the suspension until a set value hadbeen reached. This operation protocol corresponds to the cell disruptionequipment illustrated in FIG. 1(b). With the rotational speed of theSILVERSON MIXER set to 3,000 rpm and the pH setting of Lab Controlleraligned to 11.8, stirring was continued for 2 hours (40 ml of 1 Naqueous solution of sodium hydroxide was required during this time). Thetreated suspension was centrifuged (3,000 rpm, 10 min) to give aprecipitate. The precipitate was washed once with water and twice withmethanol and dried under reduced pressure to recover a powder ofp(3HB-co-3HH). The purity of this p(3HB-co-3HH) powder was as high as92% and the average molecular weight of the product polymer was 870,000.

EXAMPLE 2

Except that the pH of the suspension was adjusted with an aqueoussolution of sodium carbonate and the pH was set to 11.0, the procedureof Example 1 was otherwise repeated. The purity of the p(3HB-co-3HH)powder thus obtained was as high as 91% and the average molecular weightof the polymer was 890,000.

EXAMPLE 3

The Lab Controller described above was set to pH 11.8 as in Example 1and stirring was carried out for 1 hour. The treated suspension was fedto Manton-Gaulin manufactured by APV Gaulin, Germany, and a physicaldisruption treatment was further carried out at 7,000 psi. After thistreatment, the suspension was centrifuged (3,000 rpm, 10 min) to give aprecipitate. This precipitate was washed once with water and twice withmethanol and dried under reduced pressure to give a powder ofp(3HB-co-3HH). This p(3HB-co-3HH) powder was exceptionally pure, i.e.99%, and the average molecular weight of the polymer was 870,000.

COMPARATIVE EXAMPLE 1

Except that a mechanical stirrer (100 rpm) was used in lieu of SILVERSONMIXER for stirring, the procedure of Example 1 was otherwise repeated.The mechanical stirrer mentioned above is merely capable of stirring thesuspension and not capable of physical disruption, with the result thataddition of an alkali resulted in a viscosity elevation of thesuspension to interfere with stirring and, thus, prevented accurate pHmeasurement. The suspension was centrifuged (15,000 rpm, 10 min) but noprecipitate could be obtained.

COMPARATIVE EXAMPLE 2

Except that the pH control using Lab Controller was not carried out and1 N aqueous solution of sodium hydroxide (40 ml) was added all at once,followed by 2 hours of stirring with SILVERSON MIXER, the procedure ofExample 1 was otherwise repeated. As a result, the purity of thep(3HB-co-3HH) powder obtained was as high as 90% but the averagemolecular weight of the polymer was 300,000, indicating a markeddecrease in molecular weight.

COMPARATIVE EXAMPLE 3

An alkali treatment was carried out under the conditions described inthe best mode section of Japanese Kokai Publication Hei-07-31487. Thus,using the p(3HB-co-3HH)-containing bacterial cells, 500 ml of a cellsuspension of 40 g/L concentration was prepared. Then, the pH of thesuspension was adjusted to either 4 mM or 8 mM with 0.1 M aqueoussolution of sodium hydroxide, and the suspension was stirred at 80° C.for 1 hour with heating. Each treated suspension was cooled to roomtemperature and centrifuged for separating a precipitate but noprecipitate was obtained at the same rotational speed of 2700 rpm asused in the best mode section. Therefore, the suspension was dilutedwith equal volume of methanol and centrifuged at 8,000 rpm for 30minutes to prepare a precipitate. This precipitate was washed once withwater and twice with methanol and dried under reduced pressure to give apowder of p(3HB-co-3HH). In each case of 4 mM or 8 mM alkaliconcentration, the purity of the p(3HB-co-3HH) powder was 72% and 70%respectively, and the average molecular weights of the polymers were870,000 and 650,000, respectively. It was, therefore, clear that thepurity of the p(3HB-co-3HH) powder obtainable by this method is low andthat the decrease in polymer molecular weight is remarkable in the caseof 8 mM.

INDUSTRIAL APPLICABILITY

The method of the present invention for separating and purifying PHA canprovide PHA of high purity according to a very simple separation andpurification protocol. By the method of the invention, the pH of thesuspension can be controlled with high precision and PHA of high puritycan be obtained with good efficiency without incurring a seriousdecrease in molecular weight of the product PHA. Therefore, theinvention contributes a great deal to improved efficiency and costreduction of the commercial production of PHA by means ofmicroorganisms.

1. A method of producing a poly-3-hydroxyalkanoioc acid, which comprisescarrying out a physical disruption treatment of a suspension ofpoly-3-hydroxyalkanoic acid-containing microbial cells with adding analkali thereto either continuously or intermittently and, thereafter,separating the poly-3-hydroxyalkanoic acid.
 2. The method according toclaim 1, wherein said addition of an alkali is carried out withcontrolling the pH of the suspension.
 3. The method according to claim2, wherein the pH of the suspension is controlled between 9 and 13.5. 4.The method according to claim 1, wherein said physical disruptiontreatment is carried out under stirring of said suspension.
 5. Themethod according to claim 1, wherein said physical disruption treatmentis carried out at the temperature not less than 20° C. and below 40° C.6. The method according to claim 1, wherein the poly-3-hydroxylkanoicacid is a copolymer comprising of D-3-hydroxyhexanoate (3HH) and one ormore other 3-hydroxyalkanoic acids.
 7. The method according to claim 6,wherein the poly-3-hydroxyalkanoic acid is a binary copolymer comprisingof D-3-hydroxybutyrate (3HB) and D-3-hydroxyhexanoate (3HH) or a ternarycopolymer comprising of D-3-hydroxybutyrate (3HB), D-3-hydroxyvalerate(3HV), and D-3-hydroxhexanoate (3HH).
 8. The method according to claim1, wherein the poly-3-hydroxalkanoic acid-containing microbial cells arecells of Aeromonas caviae.
 9. The method according to claim 1, whereinthe poly-3-hydroxyalkanoic acid-containing microbial cells are cells ofa strain of microorganism transformed by a poly-3-hydroxyalkanoic acidsynthase group gene derived from Aeromonas caviae.